The protein kinase Ste20p is essential for mating in S. cerevisiae. It is responsive to signals from both heterotrimeric and Cdc42-like small G proteins and lies upstream of a MAP kinase cascade. We have isolated six cDNAs encoding three families of mammalian relatives of Ste20p to analyze their regulation and participation in MAP kinase cascades, to explore their other signaling capabilities, and to understand their place in the intracellular signal transduction network. The p21-activated kinases (PAKs) 1 and 2 are, in many respects, functional orthologs of Ste20p. They are regulated by Rac/Cdc42 small G proteins, stimulate the activities of stress-responsive MAP kinases, especially c-Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) isoforms, and mediate disassembly of actin cytoskeletal elements so that they can be reorganized by other molecules. Two other Ste20p relatives that we named TAO (thousand-and-one residues) 1 and TAO2 activate MEKs in stress- sensitive MAP kinase pathways, and thus appear to regulate MAP kinase pathways because they have MEKK activity. We have also studied the C.elegans TAO. Worms that express a constitutively active form of either mammalian or ceTAO in pharyngeal cells, where it is expressed, display retarded growth, altered electropharyngeograms, and other changes associated with a starved phenotype. Two newly identified Ste20p relatives are WNK1 and 2. A WNK ortholog exists in C.elegans. To study these enzymes we propose to: 1) Define the mechanism of regulation of NF-kappaB by PAK. What are the PAK substrates and how general is PAK's role in NF-kappaB regulation?. 2) Investigate mechanisms of regulation of PAK activity and function. In particular, we want to determine if a small G protein-independent mechanism for its activation exists. 3) Elucidate the mechanisms of activation of the JNK/SAPK pathway by PAK. 4) Define the domain structure, regulatory mechanisms, and substrates of TAO1 and 2. 5). Use genetics and biochemistry to study the function of TAO in C. elegans. 6) Determine the regulation and substrates of WNK 1 and 2. The Ste20p family has major roles in intracellular signaling networks through its actions on multiple protein kinase cascades as well as through its ability to impact on the actin cytoskeleton. Ste20p kinases coordinate the activities of multiple downstream signaling pathways not only because of their catalytic activities but also because of their capacities to bind to upstream and downstream components of signal transduction pathways. A detailed understanding of their regulation and biological activities will aid in elucidating mechanisms controlling a wide variety of cellular processes.